This project will focus on the cellular and serum factors that affect cholesterol transport between cells and high density lipoprotein (HDL)/ apoproteins. Several cell types will be used but study of macrophages will be emphasized. In Specific Aim 1 cellular factors affecting cholesterol efflux will be studied. The hypothesis to be tested is that the expression level of transporters and the contribution of aqueous diffusion in cholesterol donor cells together with the concentration/distribution of acceptor HDL particles optimal for these pathways determine efflux efficiency. We will quantify the contributions of aqueous diffusion and the SR-BI, ABCA1 and ABCG1 transporters to efflux from cells exposed to serum, isolated lipoproteins and reconstituted HDL. Protocols using both genetic manipulation and pharmacological inhibitors to measure pathway contributions to total efflux that were developed during the current grant cycle will be employed. We will also study if removal of the cholesterol produced by cholesteryl ester (CE) hydrolysis in macrophage foam cells occurs via specific efflux pathways and how oxidation of CE by lipoxygenases affects clearance. Serum factors that affect cholesterol flux will be considered in Specific Aim 2. These experiments will test the hypothesis that HDL remodeling by LCAT, CETP and PLTP alters the relative contribution of different pathways. We predict that there will be a reciprocal relationship between the effects of serum factors that increase small/lipid poor HDL and serum factors that increase large phospholipid-rich HDL. Specific Aim 3 is designed to demonstrate that the efflux capacity of human serum HDL depends on HDL quality and not just total HDL cholesterol. We will use the serum HDL fraction from specimens having similar total HDL-C but different efflux capacities to understand how macrophage cholesterol efflux is affected by HDL composition. In Specific Aim 4 we will use an assay we developed to simultaneously measure efflux and influx of cholesterol when macrophages are incubated with serum or lipoproteins. These assays will yield values for influx, efflux and net flux when macrophages are exposed to a variety of cholesterol acceptors. The studies proposed in Project 1 arise from close collaborations established in the past with Projects 2 and 3, and will continue to be well integrated with studies proposed in those projects. The accumulation of cholesterol-loaded macrophages in the arterial wall is an early event in formation of atherosclerotic plaques. We know this process is reduced by the presence of high circulating HDL levels but do not understand why. These studies will add to our understanding of HDL function.